γδ T-cells account for up to 10% of circulating lymphocytes and operate at the interface between innate and adaptive immunity. Four attributes of these versatile cells render them ripe for exploitation in therapies and in particular in cancer immunotherapy. First, γδ T-cells recognise genomic, metabolic and signaling perturbations associated with the transformed state [1, 2]. Second, they possess a diverse network of immune effector activities, overlapping and yet distinct to those deployed by “conventional” αβ T-cells. γδ T-cells release perforin and granzymes, express both FAS and TRAIL, engage in Fc receptor-dependent effector functions and produce a range of immunomodulatory cytokines, including tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-17. Third, γδ T-cells act as efficient antigen-presenting cells, enabling the perpetuation of immune attack through adaptive mechanisms [3]. Finally, since these cells are not HLA-restricted, they do not elicit graft versus host disease. This enhances the prospect of their future use in the allogeneic “off the shelf” setting [4].
Most circulating γδ T-cells in man display a Vγ9Vδ2 receptor that recognises non-peptide phosphoantigens (PAgs), best exemplified by IPP and its stereoisomer DMAPP (FIG. 1) [5]. Since PAgs are intermediates of mevalonate metabolism, Vγ9Vδ2 T-cells provide an innate mechanism to detect excess activity of this key metabolic pathway. Such surveillance is justified from an evolutionary standpoint since excess mevalonate pathway flux promotes cellular transformation, acting synergistically with p21Ras [6]. This reflects the fundamental role of this network in the biosynthesis of isoprenoids required for post-translational modification of several GTPases, including p21Ras, Cdc42, Rho, Rab and Rac.
Amino-bisphosphonate (NBP) drugs such as zoledronic (ZA), alendronic (AA), pamidronic (PA) and ibandronic acid (IA) exert anti-tumor activity through a combination of directly cytotoxic and immunomodulatory mechanisms [7]. A key example of the latter is the ability of these drugs to activate Vγ9Vδ2 T-cells. This results from inhibition of FPP synthase within the mevalonate pathway, leading to increased PAg accumulation (FIG. 1) [8]. Tumor cells that have been pulsed with NBPs rapidly acquire large PAg loads and thus become more sensitive to recognition by Vγ9Vδ2 T-cells [5, 9]. Exploitation of this principle provides an opportunity to enhance tumor susceptibility to γδ T-cell immunotherapy.
The clinical development of γδ T-cell immunotherapy builds on two established findings. First, in an effort to achieve in-vivo expansion of Vγ9Vδ2 T-cells, patients with diverse malignancies have been treated with ZA and low-dose IL-2. In many cases, these small studies have correlated circulating Vγ9Vδ2 T-cell numbers with retarded disease progression [10]. Second, ex-vivo expanded Vγ9Vδ2 T-cells have been tested as an autologous adoptive immunotherapy in several early phase clinical trials, involving diverse cancers including epithelial ovarian cancer (EOC) [11-13]. Although these studies have demonstrated the safety of infused γδ T-cells, clinical efficacy has been limited (even when combined with ZA). This highlights the need for better systems to expand these cells at high efficiency, yielding cells that exhibit improved anti-tumor activity.
Transforming growth factor-β (TGF-β) is a secreted protein that exists in at least three isoforms, called TGF-β1, TGF-β2 and TGF-β3. It is a cytokine that has a role in a variety of processes including proliferation and cellular differentiation, but also immunity and cancer. It is generally understood that in this context, it has a regulatory immune effect, and this may explain in part why it is upregulated in certain cancers, which overexpress the cytokine to reduce the host immune response. There are many papers showing that addition of TGF-β to T-cells promotes a regulatory phenotype. For example, two independent groups have shown that culturing human peripheral blood mononuclear cells (PBMCs) in the presence of cytokines that included TGF-β resulted in the production of regulatory γδ T-cells expressing high levels of Foxp3 and CD25 having an immunosuppressive function [14, 15].
The applicants have carried out studies of various protocols for the expansion of γδ T-cells and have found a particular set of conditions which produce high levels of cells with enhanced effector activity.